Use of a small native peptide activator of serca pump for treatment of heart failure and other disorders characterized by cytosolic calcium overload

ABSTRACT

The present disclosure describes a new native peptide designated herein as Dwarf Open Reading Frame, or DWORF. This peptide enhances the apparent activity of the SERCA pump, is positively inotropic and lusitropic, and therefore is provided as a therapeutic agent for disorders characterized by cytosolic calcium overload.

This application is a continuation of U.S. application Ser. No.15/491,057, filed Apr. 19, 2017, which claims benefit of priority toU.S. Provisional Application Ser. No. 62/324,706, filed Apr. 19, 2016,the entire contents of each of which are hereby incorporated byreference.

FEDERAL SUPPORT CLAUSE

This invention was made with government support under grant nos. R01HL077439-10 and R01 DK099653-01 awarded by National Institutes ofHealth. The government has certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSDP2982USC1.txt”, which is 11 KB (as measured in Microsoft Windows®)and was created on Jan. 14, 2020, is filed herewith by electronicsubmission and is incorporated by reference herein.

BACKGROUND 1. Field

The present disclosure relates generally to the fields of molecularbiology, cell physiology and medicine. More particularly, it concernsthe identification of a native small peptide that enhances the activityof the sarco/endoplasmic reticulum calcium ATPase, also known as SERCA.

2. Description of Related Art

Intracellular Ca²⁺ cycling is vitally important to the function ofstriated muscles and is altered in many muscle diseases. Upon electricalstimulation of the myocyte plasma membrane, Ca²⁺ is released from thesarcoplasmic reticulum (SR) and binds to the contractile apparatustriggering muscle contraction (Bers, 2002). Relaxation occurs when Ca²⁺is pumped back into the sarcoplasmic reticulem (SR) by thesarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA). SERCA activity isinhibited by the small transmembrane peptides phospholamban (PLN),sarcolipin (SLN), and myoregulin (MLN) in vertebrates and by sarcolambanA and B (sclA and sclB) in invertebrates, which diminish SR Ca²⁺ uptakeand myocyte contractility (Bers, 2002; MacLennan et al., 2003; Kraniasand Hajjar, 2012; Anderson et al., 2015; Bal et al., 2012; Magny et al.,2013 and Dorn, 2004).

Defective intracellular calcium homeostasis is a hallmark of cardiacdysfunction, especially with regard to calcium reuptake and cyclingduring muscle contraction, but there are no treatments currentlyavailable that effectively enhance this pathway. Directly overexpressingthe SERCA pump has proven difficult because of its large size andrequirement of post-translational modification for function. Therefore,new methods of intervening in cytosolic Ca²⁺ overload disorders isclearly needed.

SUMMARY

Thus, in accordance with the present disclosure, there is provided amethod of promoting the activity of the SERCA calcium pump in a cellcomprising contacting said SERCA pump with DWORF. DWORF may have thesequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.DWORF may be linked to apeptide or polypeptide segment, such as a cellpermeability peptide, such as HIV TAT. DWORF may be as a peptide orpolypeptide, or by expression of a nucleic acid segment coding for DWORFor a functional fragment thereof, said nucleic acid segment being underthe control of a promoter active in eukaryotic cells, such as where thenucleic acid segment is provided as naked DNA or modified mRNA, or isprovided in a viral particle, or is provided as a non-viral expressionconstruct in a nanoparticle, microparticle or lipid vehicle. The methodmay further comprise contacting the SERCA calcium pump with a secondSERCA activating agent, such as istaroxime.

The cell may be located in a living mammal, such as a non-human mammal,or a human. The contacting may occur at least a second time, such as ona chronic basis. DWORF may be provided to said mammal intravenously,intradermally, intraarterially, intraperitoneally, intranasally,topically, intramuscularly, subcutaneously, mucosally,intrapericardially, intraumbilically, orally, via injection, viainfusion, via continuous infusion, via a catheter, via a lavage, increams, or in lipid compositions (e.g., liposomes). The mammal maysuffer from a disorder characterized by or comprised of cytosoliccalcium overload, such as heart failure, restenosis or musculardystrophy. In such case, the method may further comprise administeringto said mammal a second therapy for heart failure, restenosis ormuscular dystrophy.

Also provide is an isolated polypeptide comprising a sequence selectedfrom the group consisting of wherein DWORF has the sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9. Theisolated polypeptide may be disposed in a pharmaceutically acceptablebuffer, diluent or excipient. The isolated polypeptide may consist orconsist essentially of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7 and SEQ ID NO: 9. The isolated polypeptide may be linked to aheterologous peptide or polypeptide segment.

A further embodiment provides an isolated nucleic acid segment encodinga polypeptide comprising a sequence selected from the group consistingof wherein DWORF has the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7 and SEQ ID NO: 9. The isolated nucleic acid segmentmay be disposed in a pharmaceutically acceptable buffer, diluent orexcipient. The isolated nucleic acid segment may encode a polypeptidethat consists or consists essentially of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9. The isolated nucleic acidsegment may be linked to a heterologous nucleic acid segment, such asone that encodes a cell permeability peptide, such as HIV TAT, apromoter, an expression construct, such as a viral expression construct(e.g., an adenovirus construct, a retrovirus construct, a pox virusconstruct, or a herpesvirus construct), or a non-viral expressionconstruct.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of thedisclosure, and vice versa. Furthermore, compositions and kits of thedisclosure can be used to achieve methods of the disclosure.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-I. Muscle-specific expression of DWORF. (FIG. 1A) Amino acidsequence alignment of vertebrate DWORF proteins. (FIG. 1B) Northern blotof adult mouse tissues showing DWORF RNA expression. (FIG. 1C) Detectionof DWORF RNA by qPCR in adult mouse tissues. (FIG. 1D) Detection ofDWORF RNA by qPCR in hearts of mice at the indicated ages. (FIG. 1E) The5′ UTR and the first thirteen codons of DWORF were cloned as an in-framefusion with the HaloTag protein. The empty HaloTag vector lacks aninitiation codon and is not translated. The DWORF 5′ UTR is capable ofinitiating translation and can be detected by immunoblotting with anantibody for the HaloTag protein or an antibody against DWORF. (FIG. 1F)Western blot of adult mouse tissues with the DWORF antibody revealed asingle band at the predicted size of 3.8 kDa. (FIG. 1G) Detection ofDWORF RNA by qPCR in six month old WT and aMHC-calcineurin mice. (FIG.1H) Western blot analysis of heart homogenates from WT andaMHC-calcineurin mice immunoblotted with DWORF antibody. (FIG. 1I) qPCRanalysis of human ischemic heart failure tissue showing reduced DWORFmRNA in failing hearts while atrial natriuretic peptide (NPPA) issignificantly increased.

FIGS. 2A-E. SR localization and association of DWORF with SERCA. (FIG.2A) Two-photon scanning confocal microscopy of the flexor digitorumbrevis muscle of adult mice after in vivo electroporation of plasmidsencoding GFP-DWORF, GFP-PLN or GFP-SLN indicates SR localization ofDWORF that mimics that of PLN and SLN. (M, M-line; Z, Z-line). (FIG. 2B)Co-localization of GFP-DWORF and mCherry-SERCA in transfected COS7cells. (FIG. 2C) Co-immunoprecipitation experiments in transfected COS7cells using GFP-DWORF and Myc-tagged SERCA isoforms. IP,Immunoprecipitation. (FIG. 2D) Immunoprecipitation of Myc-SERCA fromlysates of COS7 cells transfected with equal amounts of HA-DWORF, -PLN,-SLN, or -MLN and Myc-SERCA with 5-fold overexpression of either GFP orGFP-DWORF. Co-expression of GFP-DWORF reduced the pulldown of HA-taggedpeptides in association with SERCA indicating that DWORF binding toSERCA excludes binding of PLN, SLN or MLN. (FIG. 2E) Immunoprecipitationof Myc-SERCA in COST cells transfected with varying ratios of GFP-DWORFand GFP-PLN and detection with an antibody for GFP indicates that DWORFand PLN have similar binding affinities for SERCA.

FIGS. 3A-K. Consequences of DWORF gain and loss of function. (FIG. 3A) ACRISPR gRNA was generated to target the coding sequence of exon two. Anallele containing a 2-bp insertion was chosen for further experiments.The mutation is expected to produce a truncated protein lacking thetransmembrane domain. (FIG. 3B) Western blot showing the absence ofDWORF protein in the cardiac ventricle and soleus muscle of DWORFknockout (KO) mice. (FIG. 3C) Representative Ca²⁺ transients and SR loadmeasurements recorded in fluo-4 loaded cardiomyocytes from WT,α-MHC-DWORF (Tg) and DWORF KO mice. (FIG. 3D) Mean amplitude ofpacing-induced Ca²⁺ transients in fluo-4 loaded cardiomyocytes from WT,Tg and KO mice and caffeine-induced Ca²⁺ transients triggered by rapidapplication of 10 mM caffeine to quantify SR load. Ca²⁺ signal is shownas fluorescence ratio (F/F₀) with the fluorescence intensity (F)normalized to the minimal intensity measured between 0.5 Hz contractionsat diastolic phase (F₀). (FIG. 3E) Average decay time constants (Tau) ofpacing-induced Ca²⁺ transients in WT, Tg, and DWORF KO cardiomyocytesmeasured by fitting a single exponential to the Ca²⁺ transient decaytrace. This parameter is indicative of SERCA activity. (FIG. 3F) Meanvalues of decay time constants (Tau) of caffeine-induced Ca²⁺ transientsas a measure of Na⁺/Ca²⁺ exchanger (NCX) activity. (FIG. 3G)Representative pacing-induced fractional shortening traces in isolatedcardiomyocytes stimulated at 0.5 Hz as measured by edge detection. Peakfractional shortening amplitude (FIG. 3H), peak systolic Ca²⁺ transientamplitude (FIG. 3I), and systolic Ca²⁺ transient decay rates (Tau) (J)of WT, Tg and KO mice measured at baseline and in response to 10 nMisoproterenol (Iso). (FIG. 3K) Isometric force was measured from soleusmuscles mounted ex vivo and stimulated by 0.2 msec current pulsesapplied at a range of frequencies. Left: Force decay was slower in DWORFKO muscles (arrow) after fully fused tetanic contractions as shown for90 Hz (inset). Right: Slower relaxation for DWORF KO muscles occurredfor stimulus frequencies sufficient to produce twitch fusion (>20 Hz),however, unfused twitches at low frequency showed no difference inrelaxation rates. P-value<0.05, n=6.

FIGS. 4A-C. Effect of DWORF on SERCA activity measured in Ca²⁺-dependentCa²⁺-uptake assays and working model. (FIG. 4A) Ca²⁺-dependentCa²⁺-uptake assays were performed using total homogenates from hearts ofWT, α-MHC-DWORF (Tg) and DWORF KO mice to directly measure SERCAaffinity for Ca²⁺ (K_(Ca)) and SERCA activity. Mean K_(Ca) values fromn=8 hearts of each genotype are represented as bar graphs. (FIG. 4B)Ca²⁺-dependent Ca²⁺-uptake assays were performed using total homogenatesfrom soleus muscles of WT and DWORF KO mice. Mean K_(Ca) values from n=8mice of each genotype are represented as bar graphs. (C) Myocytesrelease Ca²⁺ from the SR through the ryanodine receptor (RyR), whichcauses sarcomere contraction. For muscle relaxation, Ca²⁺ must betransported back to the SR by SERCA. SERCA is inhibited by PLN. PLNinhibition is opposed by the small transmembrane peptide, DWORF, whichincreases activity of SERCA.

FIG. 5. Dworf cDNA sequence from mouse. Nucleotide sequence of a clonedfragment of Dworf from mouse heart cDNA. The ORF is highlighted in redwith the amino acid sequence below.

FIGS. 6A-B. Overview of the Dworf locus. (FIG. 6A) In mice, Dworf istranscribed from an unannotated 2.8 kb locus on chromosome 3 to producetwo transcript isoforms of approximately 300 bp that only differ byinclusion of three additional base pairs, producing a polyadenylatedRNA. The predicted open reading frame (highlighted in red) begins inexon one and ends near the 3′ end of exon two. In humans the transcriptis annotated as a lncRNA named LOC100507537 and appears to only producea single isoform. (FIG. 6B) PhyloCSF plot of the Dworf locus asextracted and analyzed from 14 different mammalian species.

FIG. 7. DWORF binding to SERCA displaces PLN in a dose-dependent manner.Immunoprecipitation of Myc-SERCA from lysates of COS7 cellsco-transfected with equal amounts of HA-PLN and Myc-SERCA and increasingamounts of either GFP-PLN or GFP-DWORF. Western blots on input samplesand bound immunoprecipitated fractions reveal that DWORF binding toSERCA competitively displaces PLN from SERCA. GFP-PLN is used as apositive control for HA-PLN displacement.

FIGS. 8A-C. Western blot analysis of heart tissue homogenates fromaMHC-DWORF transgenic mice. (FIG. 8A) Extensive western blotting ofheart homogenates from aMHC-DWORF transgenic mice reveals no significantchanges in total expression or phosphorylation status of relevant Ca²⁺handling proteins as compared to wild-type mice. A second aMHC-DWORFtransgenic line was also generated and is characterized in FIGS. 9A-C.(FIG. 8B) Immunoblots were quantified using ImageJ. Total proteinwesterns (RyR, LTCC, PMCA, SERCA, NCX and PLN) were normalized to GAPDH.(FIG. 8C) Phosphorylation blots (PS16 and PT17) were normalized to totalPLN. RyR (ryanodine receptor 2), LTCC (α1C-subunit of the voltageregulated L-Type Ca²⁺ channel), PMCA (plasma membrane Ca²⁺-ATPase),SERCA2 (SR Ca²⁺-ATPase 2), NCX (Na⁺/Ca²⁺-exchanger), PLN(phospholamban), PS16 (phospho-serine 16 on PLN), PT17(phospho-threonine 17 on PLN), GAPDH (Glyceraldehyde 3-phosphatedehydrogenase).

FIGS. 9A-C. RT-PCR and Western analysis of heart and skeletal musclefrom DWORF knockout mice. (FIG. 9A) DWORF mRNA is increasedapproximately four fold in the hearts of adult knockout mice, but othernotable genes are not altered. *P-value=0.006. (FIGS. 9B-C) Extensivewestern blotting of Ca²⁺ regulatory proteins in heart (FIG. 9B) andsoleus muscle (FIG. 9C) homogenates from DWORF KO mice reveals nosignificant changes in total protein expression levels orphosphorylation status of relevant Ca²⁺ handling proteins as compared toWT mice. Immunoblots were quantified using ImageJ. Westerns for totalprotein (RyR, LTCC, PMCA, SERCA, NCX and PLN) were normalized to GAPDHand phosphorylation blots (PS16 and PT17) were normalized to total PLN.RyR (ryanodine receptor 2), LTCC (α1C-subunit of the voltage regulatedL-Type Ca²⁺ channel), PMCA (plasma membrane Ca²⁺-ATPase), SERCA2 (SRCa²⁺-ATPase 2), NCX (Na⁺/Ca²⁺-exchanger), PLN (phospholamban), PS16(phospho-serine 16 on PLN), PT17 (phospho-threonine on PLN), GAPDH(Glyceraldehyde 3-phosphate dehydrogenase).

FIGS. 10A-C. Ca²⁺-dependent Ca²⁺-uptake assay in hearts and quadricepsmuscles from aMHC-DWORF and DWORF KO mice. (FIG. 10A) A secondaMHC-DWORF transgenic line (Tg Line 2) was generated with a more modestlevel of DWORF overexpression as assessed by DWORF immunoblotting ofheart lysates. (FIG. 10B) SERCA activity was measured using theCa²⁺-dependent Ca²⁺-uptake assay. This second transgenic line exhibiteda leftward shift of the Ca²⁺ affinity curve confirming thatoverexpression of DWORF results in elevated SERCA activity and a higheraffinity for Ca²⁺. Similar to the inventors' findings in their firsttransgenic line, modulating DWORF expression has no apparent effect onV_(max). (FIG. 10C) Ca²⁺-dependent Ca²⁺-uptake assays measuring SERCAaffinity for Ca²⁺ was unchanged in quadriceps muscle of DWORF KO.

FIGS. 11A-B. Sequences for Mouse DWORF. (FIG. 11A) Depiction of splicevariation and SNP found in DWORF protein sequences. (FIG. 11B) DWORFnucleotide sequences.

FIG. 12. Sequences for Human DWORF.

FIGS. 13A-C. DWORF overexpression rescues in vivo cardiac function inthe muscle LIM protein (MLP) KO mouse model of dilated cardiomypathy.Transthoracic echocardiographic measurements of unanesthetized mice withthe indicated genotypes. Ejection Fraction (FIG. 13A) and FractionalShortening (FIG. 13B) parameters were calculated from M-modeelectrocardiographic tracings (FIG. 13C) (* vs WT, # vs MLP KO).

FIGS. 14A-C. DWORF overexpression rescues pathological cardiacremodeling in MLP KO mice. Left ventricular (LV) dimesions werecalculated from M-mode echochardiographic measurements and are presentedas values during systole (peak contraction, LVIS, FIG. 14A) and diastole(relaxation, LVID, FIG. 14B). Heart weight to tibia length measurementswere calculated from isolated tissues (FIG. 14C) and indicate that DWORFoverexpression in MLP KO mice rescues the pathological remodeling seenin these animals (* vs WT, # vs MLP KO).

FIGS. 15A-B. DWORF overexpression rescues the ultrastructural defectsand fibrotic phenotype of MLP KO mice. (FIG. 15A) Electron microscopicanalysis of MLP KO hearts indicates severe myofibrilar disarray which isprevented by the overexpression of DWORF. (FIG. 15B) MLP KO heartsexhibit diffuse fibrosis (red color, picrosirium red stain) that isrescued when DWORF is overexpressed in the heart (DWORF Tg) of theseanimals.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, there has been significant interest in increasingthe activity of the SERCA calcium pump in the context of heart failurewhere decreased calcium release and resequestration weaken the abilityof the heart to preserve contractile function and maintain adequatetissue perfusion and eventually results in disease progression andpremature death. In rodent and large animal models of heart failure,over-expression of SERCA improves strength and endurance of the heartand ameliorates disease progression. For this reason, human clinicaltrials, named CUPID I and CUPID 2, were undertaken to evaluate thetherapeutic potential of over-expressing SERCA in heart failure patientsby viral gene delivery. Unfortunately, this trial was very recentlyreported to have failed in meeting its endpoints. Despite theunfortunate failure of this trial, there is still significant interestin boosting SERCA activity, though a final report on the trials is yetto be reported.

Here, the inventors report the isolation of a coding sequence for anendogenous transmembrane protein that enhances the apparent activity ofthe SERCA pump. Through interaction with the SERCA pump, this peptide(henceforth referred to as Dwarf Open Reading Frame or DWORF), increasesmuscle contractility and the rate of muscle relaxation, i.e., thepeptide is positively inotropic and lusitropic. SERCA function is oftendysregulated in heart failure leading to loss of contractility,relaxation, and cytosolic calcium overload. Because DWORF enhancescalcium cycling kinetics, it is an attractive candidate for genetherapies that target calcium storage and clearance. Also, because DWORFis a very small peptide, only 34 amino acids, it may be more amenable todelivery methods with restricted payload capacity.

DWORF is an endogenous enhancer of SERCA calcium pump activity, adesirable drug target for regulation of cardiac contractility. DWORF isalso an unusually small protein, which may facilitate delivery of thegene or protein to target tissues. Because DWORF is an endogenousprotein, expression of DWORF in humans would not be immunogenic,allowing for long-term dosing and expression. Previous strategies totarget the calcium handling machinery of the heart have focused onoverexpression of the SERCA pump, which is decreased in heart failure.As outlined below, DWORF presents an attractive alternative therapeuticstrategy for a number of reasons.

DWORF may be an attractive alternative to expression of SERCA formultiple reasons. First, although SERCA protein levels are decreased inheart failure, there is also an increase in a small transmembraneinhibitor of SERCA known as phospholamban. Therefore, expressing SERCAalone may not be sufficient to overcome the increased inhibition causedby phospholamban. Since DWORF can enhance the activity of SERCA in thepresence of phospholamban, it may be more beneficial to increase theactivity of the endogenous SERCA pump by expressing DWORF rather thanthe pump itself. Second, SERCA is a very large multi-pass transmembraneprotein that is among the most abundantly expressed proteins in cardiacmyocytes. These properties may make it very difficult to overexpress theprotein, especially in cells that are already compromised, meaning thatthe therapeutic threshold for this protein may be quite high. On theother hand, DWORF is a very small protein that can be rapidly producedfrom a relatively small number of transcripts. DWORF is expressed atrelatively low levels in the human myocardium (compared to rodents inwhich it is quite high), suggesting that the therapeutic threshold wouldbe much lower than for SERCA. Lastly, it has been shown that SERCA2a(the cardiac isoform of the enzyme) requires post-translationalmodification with SUMO for full activity, a process that may be limitedby the capacity for SUMOylation rather than SERCA abundance. Ectopicexpression of DWORF could increase the activity of the available SERCAwithout the need to increase its abundance.

These and other aspects of the disclosure are discussed below.

I. SERCA

SERCA, or sarco/endoplasmic reticulum Ca²⁺-ATPase, or SR Ca²⁺-ATPase, isa calcium ATPase-type P-ATPase. SERCA resides in the sarcoplasmicreticulum (SR) within muscle cells. It is a Ca²⁺ ATPase that transfersCa²⁺ from the cytosol of the cell to the lumen of the SR at the expenseof ATP hydrolysis during muscle relaxation.

There are 3 major domains on the cytoplasmic face of SERCA: thephosphorylation and nucleotide-binding domains, which form the catalyticsite, and the actuator domain, which is involved in the transmission ofmajor conformational changes. The rate at which SERCA moves Ca²⁺ acrossthe SR membrane can be controlled by the regulatory proteinphospholamban (PLB/PLN). SERCA is normally inhibited by PLB, with whichit is closely associated. Increased β-adrenergic stimulation reduces theassociation between SERCA and PLB by the phosphorylation of PLB by PKA.When PLB is associated with SERCA, the rate of Ca²⁺ movement is reduced;upon dissociation of PLB, Ca²⁺ movement increases.

Another protein, calsequestrin, binds calcium within the SR and helps toreduce the concentration of free calcium within the SR, which assistsSERCA so that it does not have to pump against such a high concentrationgradient. The SR has a much higher concentration of Ca²⁺ (10,000×)inside when compared to the cytoplasmic Ca²⁺ concentration. SERCA2 canbe regulated by microRNAs, for instance miR-25 suppresses SERCA2 inheart failure. For experimental reasons, SERCA can be inhibited bythapsigargin and induced by istaroxime.

There are 3 major paralogs, SERCA1-3, which are expressed at variouslevels in different cell types: ATP2A1-SERCA1, ATP2A2-SERCA2 andATP2A3-SERCA3. There are additional post-translational isoforms of bothSERCA2 and SERCA3, which serve to introduce the possibility ofcell-type-specific Ca²⁺-reuptake responses as well as increasing theoverall complexity of the Ca²⁺ signaling mechanism.

II. FAILURE OF CYTOSOLIC CALCIUM CLEARANCE IN CARDIAC AND SKELETALMUSCLE DISEASES

In the heart, clearance of Ca²⁺ during diastole is essential forrelaxation and storage of Ca²⁺ for successive contractions. As such,elevated end-diastolic Ca²⁺ concentration leads to decreased cardiacperformance and is a recognized feature of most forms of diastolic heartfailure (Louch et al., 2012). Changes in expression of critical proteinsand sub-cellular structure are thought to underlie defects in diastolicCa²⁺ clearance. Loss of T-tubules (a process also known as detubulation)is a documented phenomenon in advanced heart failure (Swift et al.,2012; Song et al., 2006; Louch et al., 2006; Heinzel et al., 2008).Detubulation leads to orphaned RyRs that can become asynchronous withthe cardiac action potential because of the increased distance fromDHPRs. Loss of the dyadic micro-domain also causes alteration of Ca²⁺uptake kinetics by SERCA (Brette et al., 2005). A number of changes inCa²⁺ handling genes have also been shown to be perturbed in the settingof heart failure, including SERCA, phospholamban, and NCX (Houser etal., 2000). Increases in the ratio of phospholamban to SERCA reduce thecell's ability to re-sequester Ca²⁺ in the SR, which increases tensionof the resting fiber and reduces contractile strength in subsequentcontractions. Aberration of SERCA activity may lead to a greaterreliance on NCX for Ca²⁺ removal, but increased intracellular sodium,which also may occur in heart failure, diminishes the ability of NCX toremove Ca²⁺ the cell. Therefore, restoration of SERCA activity usingDWORF or genetic sequences encoding DWORF would have a foreseeablypositive effect on cardiac contractility and by extension, morbidity andmortality of heart disease.

Restoration of SERCA activity by relieving phospholamban inhibition ofthe pump has previously been shown to have beneficial effects oncardiovascular function in a well characterized mouse model of dilatedcardiomyopathy (DCM)(Arbor et al., 1999). Muscle LIM protein (MLP) is astructural protein involved in muscle development and structuralintegrity and a knockout mouse model of MLP leads to severe heartfailure that can be completely rescued by knocking out phospholamban,which restores SERCA activity and calcium homeostasis (Minamisawa etal., 1999). Overexpression of DWORF has the same functional effect asremoval of phospholamban, thereby presenting a very attractivetherapeutic option for heart failure treatment.

Another scenario that results in increased cytosolic Ca²⁺ retentionoccurs when blood flow is restored to a previously ischemic region ofcardiac muscle. In this setting, reperfusion paradoxically results infurther injury to the tissue, a phenomenon known as ischemia reperfusioninjury. Introduction of molecules such as DWORF or genetic sequencesencoding DWORF during arterial recanalization could hasten restorationof ionic homeostasis and reduce tissue injury.

Accumulation of cytosolic Ca²⁺ has also been implicated in some skeletalmuscle diseases, namely muscular dystrophies. It is hypothesized thatloss of dystophin or other components of the dystrophin-glycoproteincomplex results in chronic microscopic shredding of the sarcolemma orhyperactivity of stretch-activated channels. These events may allow Ca²⁺to leak into the cell and accumulate by overwhelming the Ca²⁺ clearancemachinery (Allen et al., 2010). It is thought that accumulation of Ca²⁺contributes significantly to dystrophic disease progression by promotingmyofiber necrosis (Whitehead et al., 2006). This hypothesis is supportedby the fact that overexpression of stretch-activated Ca²⁺ channels inskeletal muscle results in a dystrophic phenotype (Millay et al., 2009).Recently deregulation of Ca²⁺ by way of increased intracellular sodiumand NCX over-activity were also shown to play a role in musculardystrophy (Burr et al., 2014).

Alterations in Ca²⁺ clearance have also been suggested as a contributingmechanism in physiologic fatigue of skeletal muscles, although this isamong many other hypothesized causes (Allen et al., 2008). Recent workby Anderson et al. in which the skeletal muscle inhibitor of SERCA,myoregulin, was knocked out showed an increase in running endurance inthe KO mice (Anderson et al., 2015). This work contributes furtherevidence that Ca²⁺ handling plays a crucial role in the physiology ofmuscle fatigue. Given the importance of cytosolic Ca²⁺ clearance inskeletal muscle, it is foreseeable that DWORF or genetic sequencesencoding DWORF could be of benefit in reducing the morbidity andmortality of skeletal muscle diseases with features of cytosolic Ca²⁺retention.

Because of the involvement of SERCA in muscle diseases, it ishypothesized that increasing activity of SERCA, either by pharmacologicor gene-based therapy, might be an effective strategy. In mice,overexpression of SERCA1a using a transgene or adeno-associated virus inthe muscles of dystrophic mice improved the severity of the disease(Goonasekera et al., 2011). Similarly, overexpression of SERCA2a infailing hearts of rodents, pigs, and sheep has been shown to improveheart function (del Monte et al., 2004; del Monte et al., 2001; Schmidtet al., 2000; Miyamoto et al., 2000; Hajjar et al., 1998; Byrne et al.,2008; Kawase et al., 2008 and Sakata et al., 2007). Because of the smallsize, low molecular complexity, and positive effects on SERCA activityof the DWORF protein, it is an ideal candidate molecule for treatment ofdiseases that are characterized by increased cytosolic calcium orreduced SERCA activity.

III. DWORF PEPTIDES AND POLYPEPTIDES AND NUCLEIC ACIDS CODING THEREFOR

A. DWORF Polypeptides

The DWORF polypeptide sequence is illustrated in SEQ ID NO: 1 (mouse)and SEQ ID NO: 3 (human). The mouse DWORF coding sequence is shown asSEQ ID NO: 2 (mouse) and SEQ ID NO: 4 (human). With only 34 codons,DWORF is the third smallest full-length protein known to be encoded bythe mouse genome.

The mouse DWORF transcript is encoded by 3 exons on chromosome 3. TheORF begins in exon 1, which encodes the first four amino acids of theprotein, and the remaining protein is encoded in exon 2. Alternativeusage of two adjacent splice acceptor sequences between exons 1 and 2produces two transcripts that differ by a 3 nucleotide insertion. Basedon RNA-seq reads mapping to the exon junction, the shorter isoform of 34amino acids appears to be substantially more abundant in the heart. TheDWORF ORF is conserved to lamprey, the most distant extant vertebratespecies for which a genome sequence is currently available and the ORFscores positively by PhyloCSF. The C-terminal region is enriched inhydrophobic amino acids and is predicted to encode a tail-anchoredtransmembrane peptide. The N-terminal region is less stringentlyconserved, but most sequences (except for that of Anolis carolensis)contain multiple charged residues (primarily lysine and aspartic acid)in this region.

B. DWORF Peptides

The present disclosure contemplates the design, production and use ofvarious DWORF peptides. The structural features of these peptides are asfollows. First, the peptides may have 5 to 35 consecutive residues ofDWORF. Thus, the term “a peptide having no more than X consecutiveresidues,” even when including the term “comprising,” cannot beunderstood to comprise a greater number of consecutive DWORF residues.In general, the peptides will be 35 residues or less, again, comprisingno more than 20 consecutive residues of DWORF. The overall length may be5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 residues. Ranges ofpeptide length of 5-34/35 residues, 6-34/35 residues, 7-50 residues,7-25, residues, 5-20 residues, 6-20 residues, 7-20 residues, and 7-15residues are contemplated. The number of consecutive DWORF residues maybe 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Rangesof consecutive residues of 5-20 residues, 5-20 residues, 6-20 residues,7-20 residues and 5-15 residues, 5-15, residues, 6-15 residues or 7-15residues are contemplated.

The present disclosure may utilize an L-configuration amino acids,D-configuration amino acids, or a mixture thereof. While L-amino acidsrepresent the vast majority of amino acids found in proteins, D-aminoacids are found in some proteins produced by exotic sea-dwellingorganisms, such as cone snails. They are also abundant components of thepeptidoglycan cell walls of bacteria. D-serine may act as aneurotransmitter in the brain. The L and D convention for amino acidconfiguration refers not to the optical activity of the amino aciditself, but rather to the optical activity of the isomer ofglyceraldehyde from which that amino acid can theoretically besynthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde islevorotary).

One form of an “all-D” peptide is a retro-inverso peptide. Retro-inversomodification of naturally-occurring polypeptides involves the syntheticassemblage of amino acids with .alpha.-carbon stereochemistry oppositeto that of the corresponding L-amino acids, i.e., D-amino acids inreverse order with respect to the native peptide sequence. Aretro-inverso analogue thus has reversed termini and reversed directionof peptide bonds (NH—CO rather than CO—NH) while approximatelymaintaining the topology of the side chains as in the native peptidesequence. See U.S. Pat. No. 6,261,569, incorporated herein by reference.

As mentioned above, the present disclosure contemplates fusing orconjugating a cell delivery domain (also called a cell delivery vector,or cell transduction domain). Such domains are well known in the art andare generally characterized as short amphipathic or cationic peptidesand peptide derivatives, often containing multiple lysine and arginineresides (Fischer, 2007). Of particular interest are poly-D-Arg andpoly-D-Lys sequences (e.g., dextrorotary residues, eight residues inlength), while others are shown in Table 1, below.

Also as mentioned above, peptides modified for in vivo use by theaddition, at the amino- and/or carboxyl-terminal ends, of a blockingagent to facilitate survival of the peptide in vivo are contemplated.This can be useful in those situations in which the peptide termini tendto be degraded by proteases prior to cellular uptake. Such blockingagents can include, without limitation, additional related or unrelatedpeptide sequences that can be attached to the amino and/or carboxylterminal residues of the peptide to be administered. These agents can beadded either chemically during the synthesis of the peptide, or byrecombinant DNA technology by methods familiar in the art.Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino- and/orcarboxyl-terminal residues.

1. Synthesis

It may be advantageous to produce peptides using the solid-phasesynthetic techniques (Merrifield, 1963). Other peptide synthesistechniques are well known to those of skill in the art (Bodanszky etal., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia,1984). Appropriate protective groups for use in such syntheses will befound in the above texts, as well as in Protective Groups in OrganicChemistry, 1973. These synthetic methods involve the sequential additionof one or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using solid phase synthesis as an example, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptides of the disclosure are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

Aside from the 20 standard amino acids can be used, there are a vastnumber of “non-standard” amino acids. Two of these can be specified bythe genetic code, but are rather rare in proteins. Selenocysteine isincorporated into some proteins at a UGA codon, which is normally a stopcodon. Pyrrolysine is used by some methanogenic archaea in enzymes thatthey use to produce methane. It is coded for with the codon UAG.Examples of non-standard amino acids that are not found in proteinsinclude lanthionine, 2-aminoisobutyric acid, dehydroalanine and theneurotransmitter gamma-aminobutyric acid. Non-standard amino acids oftenoccur as intermediates in the metabolic pathways for standard aminoacids—for example ornithine and citrulline occur in the urea cycle, partof amino acid catabolism. Non-standard amino acids are usually formedthrough modifications to standard amino acids. For example, homocysteineis formed through the transsulfuration pathway or by the demethylationof methionine via the intermediate metabolite S-adenosyl methionine,while hydroxyproline is made by a posttranslational modification ofproline.

2. Linkers

Linkers or cross-linking agents may be used to fuse DWORF peptides toother proteinaceous sequences. Bifunctional cross-linking reagents havebeen extensively used for a variety of purposes including preparation ofaffinity matrices, modification and stabilization of diverse structures,identification of ligand and receptor binding sites, and structuralstudies. Homobifunctional reagents that carry two identical functionalgroups proved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described in U.S. Pat.No. 5,889,155, specifically incorporated herein by reference in itsentirety. The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides. In instances where a particular peptidedoes not contain a residue amenable for a given cross-linking reagent inits native sequence, conservative genetic or synthetic amino acidchanges in the primary sequence can be utilized.

Another use of linkers in the context of peptides as therapeutics is theso-called “Stapled Peptide” technology of Aileron Therapeutics. Thegeneral approach for “stapling” a peptide is that two key residueswithin the peptide are modified by attachment of linkers through theamino acid side chains. Once synthesized, the linkers are connectedthrough a catalyst, thereby creating a bridge the physically constrainsthe peptide into its native α-helical shape. In addition to helpingretain the native structure needed to interact with a target molecule,this conformation also provides stability against peptidases as well ascell-permeating properties. U.S. Pat. Nos. 7,192,713 and 7,183,059,describing this technology, are hereby incorporated by reference. Seealso Schafmeister et al., J. American Chemical Soc., 2000, 122(24): p.5891-5892.

C. Purification

In certain embodiments, the polypeptide and peptides of the presentdisclosure may be purified. The term “purified,” as used herein, isintended to refer to a composition, isolatable from other components,wherein the protein is purified to any degree relative to itsnaturally-obtainable state. A purified protein therefore also refers toa protein, free from the environment in which it may naturally occur.Where the term “substantially purified” is used, this designation willrefer to a composition in which the protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the proteins inthe composition.

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. Other methods for protein purification include,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; gel filtration, reversephase, hydroxylapatite and affinity chromatography; and combinations ofsuch and other techniques.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. Another method forassessing the purity of a fraction is to calculate the specific activityof the fraction, to compare it to the specific activity of the initialextract, and to thus calculate the degree of purity. The actual unitsused to represent the amount of activity will, of course, be dependentupon the particular assay technique chosen to follow the purificationand whether or not the expressed protein or peptide exhibits adetectable activity.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE. It will thereforebe appreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified expressionproducts may vary.

D. Nucleic Acids and Expression

In certain embodiments, DWORF may be delivered to a SERCA pump or asubject via nucleic acid that is expressed in a eukaryotic expressionsystem. Expression cassettes are employed to express DWORF afterdelivery to a cell/subject, i.e., for use directly in a genetic-baseddelivery approach, and also for in vitro synthesis and subsequentpurification of the protein. Expression requires that appropriatesignals be provided in the vectors, and include various regulatoryelements such as enhancers/promoters from both viral and mammaliansources that drive expression of the genes of interest in cells.Elements designed to optimize messenger RNA stability andtranslatability in host cells also are defined. The conditions for theuse of a number of dominant drug selection markers for establishingpermanent, stable cell clones expressing the products are also provided,as is an element that links expression of the drug selection markers toexpression of the polypeptide.

Nucleic acids according to the present disclosure may encode all ofDWORF, a domain of DWORF that stimulates SERCA, or any other fragment ofDWORF. The nucleic acid may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In preferredembodiments, however, the nucleic acid would comprise complementary DNA(cDNA). Also contemplated is a cDNA plus a natural intron or an intronderived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and other nucleic acidsof the present disclosure may be used as molecular weight standards in,for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a given DWORF from a given species may berepresented by natural variants that have slightly different nucleicacid sequences but, nonetheless, encode the same protein (see discussionin the Examples, below).

As used in this application, the term “a nucleic acid encoding a DWORF”refers to a nucleic acid molecule that has been isolated free of totalcellular nucleic acid. In certain embodiments, the disclosure concerns anucleic acid sequence essentially as set forth in the sequence listing.The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids, as discussed in the following pages.

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 80%, preferably at least about90% and most preferably about 95% of nucleotides that are identical tothe nucleotides of the sequences set forth in the sequence listing.Sequences that are essentially the same as those set forth in sequencelisting also may be functionally defined as sequences that are capableof hybridizing to a nucleic acid segment containing the complement ofthe sequences set forth in the sequence listing under standardconditions.

The DNA segments of the present disclosure include those encodingbiologically functional equivalent DWORF proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

1. Regulatory Elements

Throughout this application, the term “expression cassette” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed and translated, i.e., is underthe control of a promoter. A “promoter” refers to a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa gene. The phrase “under transcriptional control” means that thepromoter is in the correct location and orientation in relation to thenucleic acid to control RNA polymerase initiation and expression of thegene. An “expression vector” is meant to include expression cassettescomprised in a genetic construct that is capable of replication, andthus including one or more of origins of replication, transcriptiontermination signals, poly-A regions, selectable markers, andmultipurpose cloning sites.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

In certain embodiments, viral promotes such as the human cytomegalovirus(CMV) immediate early gene promoter, the SV40 early promoter, the Roussarcoma virus long terminal repeat, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized. Further, selection of a promoter that is regulated inresponse to specific physiologic signals can permit inducible expressionof the gene product.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Below is a list of promoters/enhancers and inducible promoters/enhancersthat could be used in combination with the nucleic acid encoding a geneof interest in an expression construct (Table 2 and Table 3).Additionally, any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression of thegene. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct.

TABLE 2 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Banerji et al., 1983; Gilles et al., 1983; Heavy ChainGrosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Queen et al., 1983; Picard et al., 1984 Light ChainT-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or Sullivan et al., 1987 DQ β β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Greene et al., 1989; Lin et al., 1990Receptor MHC Class II 5 Koch et al., 1989 MHC Class II Sherman et al.,1989 HLA-DRa β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Jaynes et al., 1988; Horlick et al., 1989; Johnson Kinase (MCK)et al., 1989 Prealbumin Costa et al., 1988 (Transthyretin) Elastase IOrnitz et al., 1987 Metallothionein Karin et al., 1987; Culotta et al.,1989 (MTII) Collagenase Pinkert et al., 1987; Angel et al., 1987aAlbumin Pinkert et al., 1987; Tronche et al., 1989, 1990 α-FetoproteinGodbout et al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Hirsh et al., 1990 Adhesion Molecule (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Ripe et al., 1989 Type I Collagen Glucose-RegulatedChang et al., 1989 Proteins (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Edbrooke et al., 1989 Amyloid A (SAA) TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Pech et al., 1989 GrowthFactor (PDGF) Duchenne Muscular Klamut et al., 1990 Dystrophy SV40Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak etal., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986;Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner etal., 1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980;Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983;de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988;Campbell and Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human Muesing et al.,1987; Hauber et al., 1988; Immunodeficiency Jakobovits et al., 1988;Feng et al., 1988; Virus Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Cytomegalovirus Weber et al., 1984; Boshart et al., 1985;Foecking (CMV) et al., 1986 Gibbon Ape Holbrook et al., 1987; Quinn etal., 1989 Leukemia Virus

TABLE 3 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; Lee et mammary al., 1981; Majors etal., tumor virus) 1983; Chandler et al., 1983; Ponta et al., 1985; Sakaiet al., 1988 β-Interferon poly(rI)x Tavernier et al., 1983 poly(rc)Adenovirus 5 E2 ElA Imperiale et al., 1984 Collagenase Phorbol Ester(TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al.,1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX GeneInterferon, Hug et al., 1988 Newcastle Disease Virus GRP78 Gene A23187Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989 VimentinSerum Rittling et al., 1989 MHC Class I Gene Interferon Blanar et al.,1989 H-2κb HSP70 ElA, SV40 Large Taylor et al., 1989, 1990a, T Antigen1990b Proliferin Phorbol Ester-TPA Mordacq and et al., 1989 TumorNecrosis PMA Hensel et al., 1989 Factor Thyroid Thyroid HormoneChatterjee et al., 1989 Stimulating Hormone α Gene

Of particular interest are muscle specific promoters, and moreparticularly, cardiac specific promoters. These include the myosin lightchain-2 promoter (Franz et al., 1994; Kelly et al., 1995), the α-actinpromoter (Moss et al., 1996), the troponin 1 promoter (Bhaysar et al.,1996); the Na⁺/Ca²⁺ exchanger promoter (Barnes et al., 1997), thedystrophin promoter (Kimura et al., 1997), the α7 integrin promoter(Ziober and Kramer, 1996), the brain natriuretic peptide promoter(LaPointe et al., 1996) and the αB-crystallin/small heat shock proteinpromoter (Gopal-Srivastava, 1995), α-myosin heavy chain promoter(Yamauchi-Takihara et al., 1989) and the ANF promoter (LaPointe et al.,1988).

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the disclosure, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

2. Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the disclosure, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kB of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In one system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Racher et al. (1995) disclosed improved methods for culturing 293 cellsand propagating adenovirus. In one format, natural cell aggregates aregrown by inoculating individual cells into 1 liter siliconized spinnerflasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the disclosure. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent disclosure. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present disclosureis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to thedisclosure. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors, asdescribed by Karlsson et al. (1986), or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹² plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present disclosure. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

Other viral vectors may be employed as expression constructs in thepresent disclosure. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the presentdisclosure. These include calcium phosphate precipitation (Graham andVan Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer etal., 1987), gene bombardment using high velocity microprojectiles (Yanget al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wuand Wu, 1988). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the disclosure, the expression constructmay simply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the disclosure for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentdisclosure.

In a further embodiment of the disclosure, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection. A reagent known as Lipofectamine 2000™ iswidely used and commercially available.

In certain embodiments of the disclosure, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentdisclosure. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

3. Modified mRNAs

Another approach for gene expression employs synthetic modified RNAsequences, and in particular for in vivo protein expression in tissuesand organs, and also in cells in vivo, e.g., muscle cells including butnot limited to cardiomyocytes and myogenic cells. Other aspects relateto use of modified-RNAs encoding DWORF for treatment of diseases anddisorders, for example, but not limited to muscle disorders andcardiovascular diseases and disorders in a subject. Other aspects relateto pharmaceutical compositions and kits thereof comprising a least onesynthetic modified-RNA encoding DWORF for administration to a subjectfor treatment of diseases or disorders in a subject. In someembodiments, the disease or disorder is a cardiovascular disease ordisorder.

Recently, it has been reported that synthetic, modified RNA (hereinreferred to as “MOD-RNA”) can be used for overexpression of a gene ofinterest in mammalian cells in vitro. The chemical and sequencemodifications made in the synthetic mRNA stabilize the molecule andenhance transcription. Expression of polypeptides from MOD-RNA allowsfor highly efficient, transient expression of a gene of interest invitro without requiring introduction of DNA or viral sequences that maybe integrated into the host cell. Demonstrated delivery of syntheticmodified RNAs using tailored transfection techniques, and in someembodiments, administration of MOD-RNAs in a composition can alsocomprise specific reagents that inhibit degradation of an introduced,synthetic modified RNA. WO 2012138453 A1 provides supporting informationon such techniques.

IV. THERAPIES

A. Heart Failure and Diseases Characterized by Cytosolic Ca²⁺ Retention

There are many causes of heart failure syndromes, but they generallyresult in inability of the cardiac muscle to provide sufficientperfusion to tissues, resulting in systemic ischemia and fluidaccumulation. All heart failure patients have similar symptoms, butfailure can result from multiple etiologies, most being related toinsufficiency of contractile strength, relaxation to allow for filling,or a combination of both features. Calcium handling is vital to both ofthese etiologies of heart failure. For the scenario of impairedcontractility, internal calcium stores of the myocyte are typicallyinsufficient or release from the SR is dampened either because of damageto the microarchitecture of the myocyte or kinetic dysfunction of thecalcium storage and release machinery. Diastolic failure can havesimilar molecular origins, but ultimately results in delayed clearanceof calcium following contraction, which results in incompleterelaxation. The SERCA pump is the only route for calcium to bere-sequestered back to the SR following contraction, and dysfunctionalSERCA expression or activity has been extensively linked to heartfailure syndromes. Since DWORF modulates the activity of the SERCA pump,it would be a rational therapy for heart failure.

In addition to heart failure, DWORF is also contemplated for use indecreasing reperfusion injury following coronary artery catherization totreat a myocardial infarction. Reperfusion injury is a paradoxicalfurther injury to ischemic tissue following restoration of blood flow,i.e., reperfusion. It is thought that this phenomenon is caused byinflux of calcium and sodium during reperfusion, which then leads tohypercontraction, activation of proteolytic enzymes and metabolicstress. Administration of DWORF or its genetic code by viral or MOD-RNAduring catheterization could potentially limit the extent of this typeof very common injury.

Finally, DWORF may also used as a treatment for muscular dystrophy.Although far less common than heart failure or coronary disease, MDcauses a significant morbidity and mortality for those affected. InDuchenne muscular dystrophy models, excessive calcium influx results indamage similar to that described for reperfusion injury. It isreasonable to predict that DWORF could treat this disease.

B. Pharmaceutical Formulations and Routes of Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. In some embodiments, such formulation with thecompounds of the present disclosure is contemplated. Generally, thiswill entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

One will generally desire to employ appropriate salts and buffers torender agents stable and allow for uptake by target cells. Aqueouscompositions of the present disclosure comprise an effective amount ofthe agents to cells, dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium. Such compositions also arereferred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the agents of the present disclosure, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present disclosure may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present disclosure will be via any common route so longas the target tissue is available via that route. Such routes includeoral, nasal, buccal, rectal, vaginal or topical route. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intracardiac, subcutaneous, intramuscular, intratumoral,intraperitoneal, or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The compositions of the present disclosure may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences,” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

C. Methods of Treatment

In particular, the compositions described above are preferablyadministered to a mammal (e.g., rodent, human, non-human primates,canine, bovine, ovine, equine, feline, etc.) in an effective amount,that is, an amount capable of producing a desirable result in a treatedsubject (e.g., reducing cytosolic calcium overload). Toxicity andtherapeutic efficacy of the compositions utilized in methods of thedisclosure can be determined by standard pharmaceutical procedures. Asis well known in the medical and veterinary arts, dosage for any oneanimal depends on many factors, including the subject's size, bodysurface area, body weight, age, the particular composition to beadministered, time and route of administration, general health, theclinical symptoms of cancer and other drugs being administeredconcurrently. A composition as described herein is typicallyadministered at a dosage and route that stimulates SERCA. In someembodiments, amounts of the agents used are calculated to be from about0.01 mg to about 10,000 mg/day. In some embodiments, the amount is fromabout 1 mg to about 1,000 mg/day. In some embodiments, these dosings maybe reduced or increased based upon the biological factors of aparticular patient such as increased or decreased metabolic breakdown ofthe drug or decreased uptake.

The therapeutic methods of the disclosure (which include prophylactictreatment) in general include administration of a therapeuticallyeffective amount of the compositions described herein to a subject inneed thereof, including a mammal, particularly a human. Such treatmentwill be suitably administered to subjects, particularly humans,suffering from, having, susceptible to, or at risk for a diseaseassociated with cytosolic calcium overload, or symptom thereof.Determination of those subjects “at risk” can be made by any objectiveor subjective determination by a diagnostic test or opinion of a subjector health care provider (e.g., genetic test, enzyme or protein marker,marker (as defined herein), family history, and the like).

D. Combination Therapies

It is envisioned that the peptides/peptoids described herein may be usedin combination therapies with an additional therapeutic agent orregimen. It is very common in the field of medicine to combinetherapeutic modalities, both for increased efficacy, and reduction ofdosages and hence side effects. The following is a general discussion oftherapies that may be used in conjunction with the therapies of thepresent disclosure.

To treat diseases or disorders characterized by cytosolic calciumoverload using the methods and compositions of the present disclosure,one would generally contact a subject with a compound and at least oneother therapy. These therapies would be provided in a combined amounteffective to achieve a reduction in one or more disease parameter. Thisprocess may involve contacting the cells/subjects with the bothagents/therapies at the same time, e.g., using a single composition orpharmacological formulation that includes both agents, or by contactingthe cell/subject with two distinct compositions or formulations, at thesame time, wherein one composition includes the compound and the otherincludes the other agent.

Alternatively, the peptides/peptoids described herein may precede orfollow the other treatment by intervals ranging from minutes to weeks.One would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the therapies wouldstill be able to exert an advantageously combined effect on thecell/subject. In such instances, it is contemplated that one wouldcontact the cell with both modalities within about 12-24 hours of eachother, within about 6-12 hours of each other, or with a delay time ofonly about 1-2 hours. In some situations, it may be desirable to extendthe time period for treatment significantly; however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

It also is conceivable that more than one administration of either thecompound or the other therapy will be desired. Various combinations maybe employed, where a DWORF therapy is “A,” and the other SERCA-basedtherapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are also contemplated.

Istaroxime is an investigational drug now under development fortreatment of acute decompensated heart failure. It is still inearly-stage development, having been evaluated in phase two clinicaltrials. Istaroxime is an effective treatment for both systolic anddiastolic heart failure. Systolic heart failure is characterized byimpaired ventricular emptying, caused by reduced contractility, anddiastolic dysfunction is defined by defective ventricular filling,caused by the heart's inability to properly relax between beats.Intracellular calcium fluxes regulate both contraction and relaxation.Cardiac muscle cells from patients with heart failure show smalleramounts of peak calcium in their cytoplasm during contraction, andslower removal. The mishandling of intracellular calcium is often due toproblems in the cells' ability to mediate calcium influx, andsequestration of calcium back in the sarcoplasmic reticulum.

Istaroxime is a positive inotropic agent that mediates its actionthrough inhibition of sodium/potassium adenosine triphosphatase (Na⁺/K⁺ATPase). Na⁺/K⁺ ATPase inhibition increases intracellular sodium levels,which reverses the driving force of the sodium/calcium exchanger,inhibiting calcium extrusion and possibly facilitating calcium entry.Additionally, Istaroxime increases intracellular calcium by improvingthe efficacy by which intracellular calcium triggers sarcoplasmicreticulum calcium release, and by accelerating the inactivation state ofL-type calcium channels, which allow for calcium influx. Together thechanges in calcium handling increase cell contraction.

Istaroxime also enhances the heart's relaxation phase by increasing therate of intracellular calcium sequestration by Sarco/endoplasmicReticulum Calcium ATPase, isotype 2a (SERCA2a). SERCA2a is inhibited byphospholamban and higher phospholamban-to-SERCA2a ratios cause SERCAinhibition and impaired relaxation. Istaroxime reducesSERCA2α-phospholamban interaction, and increases SERCA2a affinity forcytosolic calcium. Studies on failing human heart tissue show thatIstaroxime increases SERCA2a activity up to 67%.

Clinical trials show that Istaroxime improves ejection fraction, strokevolume and systolic blood pressure, while also enhancing ventricularfilling. The drug also reduces heart rate and ventricular diastolicstiffness. Contrary to available inotropic therapies, Istaroxime maypermit cytosolic calcium accumulation while avoiding a proarrhythmicstate. Proposed mechanisms for Istaroxime's antiarrhythmic effectinclude a suppression of the transient inward calcium current directlyinvolved in the production of delayed after-depolarizations and improvedcalcium sequestration due to SERCA2a stimulation. SERCA down-regulationin the failing myocardium might sensitize patients to the detrimentaleffect of other currently used positive inotropes. Istaroxime'slusitropic effect facilitates its wider margin of safety, as patientscan receive higher doses without signs of arrhythmias.

Thapsigargin is non-competitive inhibitor of the sarco/endoplasmicreticulum Ca²⁺ ATPase (SERCA). Structurally, thapsigargin is classifiedas a sesquiterpene lactone, and is extracted from a plant, Thapsiagarganica. It is a tumor promoter in mammalian cells. Thapsigarginraises cytosolic (intracellular) calcium concentration by blocking theability of the cell to pump calcium into the sarcoplasmic andendoplasmic reticula. Store-depletion can secondarily activate plasmamembrane calcium channels, allowing an influx of calcium into thecytosol. Thapsigargin specifically inhibits the fusion of autophagosomeswith lysosomes; the last step in the autophagic process. The inhibitionof the autophagic process in turn induces stress on the endoplasmicreticulum which ultimately leads to cellular death.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

Example 1—Materials and Methods

Identification of Conserved Small Open Reading Frames.

Total RNA was extracted from adult mouse heart tissue using Trizol(Invitrogen). An RNA sequencing library was prepared using the TruSeqRNA Library Prep Kit (Illumina) according to the manufacturer'sprotocol. Reads were mapped to the UCSC mm9 reference genome (Kent etal., 2002) using TopHat (Trapnell et al., 2009). Final transcripts wereassembled using Cufflinks and consolidated with the mm9 referenceannotations (Trapnell et al., 2012). Novel transcripts (i.e., those notexisting in the reference annotation) were extracted and combined withtranscripts annotated as long non-coding RNA in the UCSC database.Sequence alignments from fourteen mammalian species (mouse, rabbit, rat,human, chimpanzee, rhesus monkey, shrew, dog, cat, horse, cow,armadillo, elephant, and tenrec) were extracted using the “Stitch Geneblocks” tool in Galaxy (Blankenberg et al., 2011; Giardine et al., 2005;Blankenberg et al., 2010 and Goecks et al., 2010). PhyloCSF was used tocalculate conservation scores for potential ORFs in three frames on bothstrands (Lin et al., 2011). Only the highest scoring ORF for eachtranscript was reported.

Quantitative mRNA Measurement.

Total RNA was extracted from adult mouse tissues using Trizol andreverse transcribed using the SuperScript III First-Strand SynthesisSystem (Invitrogen) with a 1:1 mix of random hexamer and oligo-dTprimers. Quantitative Polymerase Chain Reaction (qPCR) was performedusing 5′ nuclease assays on the StepOne Real-Time PCR System (LifeTechnologies). The following oligonucleotides were ordered fromIntegrated DNA Technologies to measure endogenous DWORF abundance:

Forward (SEQ ID NO: 11) 5′-TTCTTCTCCTGGTTGGATGG-3′; Reverse(SEQ ID NO: 12) 5′-TCTTCTAAATGGTGTCAGATTGAAGT-3′; Probe (SEQ ID NO: 13)5′-TTTACATTGTCTTCTTCTAGAAAAGGAAGAAG-3′.Probes were labeled with 5′ 6-FAM, an internal ZEN quencher, and a 3′Iowa Black quencher and used in a 2:1 ratio with primers. Human cDNAswere quantified using SYBR Green with the following primers:

(SEQ ID NO: 14) Forward 5′-CCACCCACCAACAGGAATA-3′; (SEQ ID NO: 15)Reverse 5′-TTATGATGCAGCCCACAATC-3′.Each sample was normalized to a Eukaryotic 18S rRNA Endogenous Control(Life Technologies) reaction.

Northern Blot Analysis.

A DWORF cDNA fragment was PCR purified from heart RNA extract with thefollowing primers:

(SEQ ID NO: 16) Forward 5′-TTTCCAAAAGATAGGAAATACTACAGC-3′;(SEQ ID NO: 17) Reverse 5′-ACTCCTGGCCCTGACTAAGC-3′.The fragment was gel purified and cloned into the TOPO pCR2.1 plasmid(Life Technologies). Following amplification in E. coli, the probetemplate was excised with EcoRI and gel purified. Radiolabeled probe wasprepared using the RadPrime DNA Labeling System (Life Technologies) withα-³²P-dCTP. Radiolabeled probe was hybridized overnight at 68° C. to acommercial northern blot (Zyagen, MN-MT-1) containing 20 μg of total RNAper sample. The hybridized blot was washed four times and then exposedto autoradiography film for twenty-four hours. The blot was thenstripped using a published protocol and probed for the 18S rRNA loadingcontrol using the same procedure described for DWORF.

Antibody Derivation.

A custom polyclonal antibody was derived against the N-terminal regionof the predicted DWORF protein by New England Peptide. Rabbits wereimmunized with a synthetic peptide with the followingsequence—MAEKESTSPHLIC (SEQ ID NO: 18). Sera were collected and affinitypurified against the peptide immunogen.

Human Heart Failure Tissue.

Samples were acquired from the Temple University human heart tissue bank(IRB #21319). All ischemic heart failure samples were core biopsies ofpost-MI heart failure patients at the time of transplant. An n of 8 wasused for both failing and non-failing hearts.

Western Blot Analysis.

Lysates were prepared by pulverizing snap frozen tissues in liquidnitrogen and then homogenizing in RIPA buffer (150 mM NaCl; 1% v/vIgepal CA-630; 50 mM Tris-Cl, pH 8.3; 0.5% w/v sodium deoxycholate; 0.1%w/v sodium dodecyl sulfate) with added protease inhibitors (cOmpleteULTRA mini tablet, Roche) on ice using a ‘tight’ glass douncehomogenizer. Protein concentrations were determined using a BCA ProteinAssay Kit (Pierce). For DWORF and PLN, samples were separated on a 16.5%tricine buffered polyacrylamide gel (BioRad). Samples for otherexperiments were separated on Any kD tris-glycine bufferedpolyacrylamide gels (BioRad). DWORF was electroblotted onto ImmobilonP^(SQ) membranes (Millipore) using a semi-dry apparatus for 30 min at 20V. Other experiments were electroblotted onto Immobilon P membranesusing a semi-dry apparatus for 50 min at 20 V. Membranes were blockedovernight at 4° C. in blotto (5% w/v non-fat dry milk in TBST). Primaryantibody hybridization was carried out overnight at 4° C. with thefollowing antibodies: DWORF, 1:5,000; total PLN (2D12, Pierce), 1:5,000;pSer¹⁶-PLN (Badrilla), 1:5,000; pThr¹⁷-PLN (Badrilla), 1:5,000; SERCA2(2A7-A1, Pierce), 1:2000; NCX1 (Abcam), 1:500; RyR2 (Pierce, C3-33),1:1000; LTCC (Millipore, α1C), 1:1000; PMCA (Pierce, 5F10), 1:250; GAPDH(Millipore); 1:10,000. Blots were washed five times for five minuteseach, in TBST and then incubated with HRP-conjugated secondaryantibodies (BioRad) at 1:20,000. Blots were then developed withchemiluminescent substrate and exposed to either autoradiograph film ora digital ChemiDoc system (G:BOX, GeneSys).

Generation of Mouse Lines.

All experiments involving animals were approved by the InstitutionalAnimal Care and Use Committee at the University of Texas SouthwesternMedical Center. Knockout mice were generated using the CRISPR/Cas9system by pronuclear and cytoplasmic injection of mouse embryos withguide RNA (gRNA) and Cas9 mRNA as described previously (Long et al.,2014). Briefly, a gRNA was cloned that targeted the predicted codingsequence of DWORF. Cleavage efficiency was tested in cell culture, andthen the gRNA and Cas9 mRNA were transcribed in vitro and spin-columnpurified. Mouse embryos were injected with an equal ratio of gRNA andCas9 mRNA into the pronucleus and cytoplasm and then transferred to asurrogate dam for gestation. Because F₀ founders are expected to bemosaic, allelic disruption was confirmed in the F₀ generation pups usingthe T7E1 endonuclease assay on tail biopsies, and positive founders werebred to wild-type C57/BL6 mice to isolate potential knockout alleles.Mutants in the F₁ generation were identified by T7E1 assay and then thealleles were cloned and sequenced. One mouse line with a two-bpinsertion was chosen for further study.

Transgenic mice were derived by pronuclear injection of mouse embryos.Briefly, the DWORF coding sequence was cloned into an αMHCpromoter-driven plasmid with a polyadenylation sequence from the humangrowth hormone (hGH) gene. The plasmid was injected into the pronucleusof mouse embryos and then implanted in a surrogate dam for gestation. F₀generation pups were selected by presence of transgene by PCR from atail biopsy and bred to wild-type. F₁ generation pups were bred toC57/BL6 mice and expression of the transgene was verified in the F2progeny by qPCR.

Genotyping of Mouse Lines.

Knockout mice were genotyped using a custom TaqMan genotyping assay(Life Technologies). Briefly, tail biopsies were digested in lysisbuffer (50 mM KCl; 10 mM Tris-C1, pH 8.3; 2.5 mM MgCl₂; 0.1 mg/mLporcine gelatin; 0.45% v/v Igepal CA-630; 0.45% v/v Tween 20) withproteinase K (6 U/mL) overnight at 55° C. Particulates were removed byhigh-speed centrifugation, and the supernatant was diluted 1:10 inwater. The tail samples were then analyzed by qPCR with a mixture of thefollowing oligonucleotides:

Forward Primer (SEQ ID NO: 19) 5′-TCATTGCTTCTAAGCAGAGTCAACA-3′;Reverse Primer (SEQ ID NO: 20) 5′-ATGCAGCCTACAATCCATCCAA-3′; WT Probe(SEQ ID NO: 21) 5′-CCAGGAGAAGAATG-3′; KO Probe (SEQ ID NO: 22)5′-CAGGAGACAAGAATG-3′.Transgenic mice were genotyped based on presence or absence of the hGHsequence. Tail biopsies were processed as described for the DWORF KOmice, and used for PCR with a 1:1:1:1 mixture of the following primers(myogenin primers are used as a positive control):

hGH Forward Primer (SEQ ID NO: 23) 5′-GTCTGACTAGGTGTCCTTCT-3′;hGH Reverse Primer (SEQ ID NO: 24) 5′-CGTCCTCCTGCTGGTATAG-3′;Myogenin Forward Primer (SEQ ID NO: 25) 5′-TTACGTCCATCGTGGACAGC-3′;Myogenin Reverse Primer (SEQ ID NO: 26) 5′-TGGGCTGGGTGTTAGCCTTA-3′.PCR products were analyzed by agarose gel electrophoresis.

Co-Immunoprecipitations (CoIPs).

CoIPs were performed as described in detail (Anderson et al., 2009). HEK293 cells or COST cells were transfected with GFP-DWORF, GFP-PLN,HA-DWORF, HA-PLN, HA-SLN, HA-MLN and Myc-tagged SERCA.Immunoprecipitations were carried out using mouse anti-GFP (LifeTechnologies) or mouse anti-Myc antibody (Invitrogen) and collected withDynabeads (Life Technologies). Standard western blot procedures wereperformed on IP fractions using HRP-conjugated GFP (Pierce, GF28R), Myc(Pierce, 9E10) or HA (Pierce, 2-2.2.14) antibodies.

Mouse Cardiomyocyte Isolation.

Adult mouse myocytes were isolated as described (Makarewich et al., 2014and Jaleel et al., 2008). Anesthesia was induced using 3% isoflurane andmaintained using 1% isoflurane. Mouse hearts were rapidly excised andthe aorta was cannulated on a constant-flow Langendorff apparatus. Theheart was digested by perfusion of Tyrode's solution containing 0.2mg/mL Liberase DH (Roche), 0.14 mg/mL Trypsin (Gibco/Invitrogen) and(mM): CaCl₂ 0.02, glucose 10, HEPES 5, KCl 5.4, MgCl₂ 1.2, NaCl 150,sodium pyruvate 2, pH 7.4. When the tissue softened, the left ventriclewas isolated and gently minced, filtered, and equilibrated in Tyrode'ssolution with 200 μM CaCl₂, and 1% bovine serum albumin (BSA) at roomtemperature.

Ca²⁺ Transients and SR Load.

Myocytes were loaded with 5 μM fluo-4 AM (Molecular Probes) and placedin a heated chamber (35° C.) on the stage of an inverted microscope andperfused with Tyrode's solution containing in mM: CaCl₂ 1, glucose 10,HEPES 5, KCl5.4, MgCl₂ 1.2, NaCl 150, sodium pyruvate 2, pH 7.4.Myocytes were paced at 0.5 Hz and fractional shortening data wascollected using edge detection. For intracellular Ca²⁺ fluorescencemeasurements, the F₀ (or F unstimulated) was measured as the averagefluorescence of the cell 50 ms prior to stimulation. The maximal fluo-4fluorescence (F) was measured at peak amplitude. Background fluorescencewas subtracted from each parameter before representing the peak Ca²⁺transient as F/F₀ (Makarewich et al., 2014 and Jaleel et al., 2008). Tau(τ) was measured as the decay rate of the average Ca²⁺ transient trace.Isoproterenol (Iso, Sigma) was used at 10 nM.

To measure SR Ca²⁺ content, myocytes were paced at 0.5 Hz for 10consecutive contractions, and 10 mM caffeine (Sigma) was then rapidlyapplied via a glass pipette close to the myocyte with a Pico spritzer(Piacentino et al., 2003). The decay of caffeine-induced Ca²⁺ transientswas fit with a single-exponential function, and time constant (τ) valuesindicate Na⁺/Ca²⁺-exchanger (NCX) activity.

Oxalate-Supported Ca²⁺ Uptake Measurements.

Oxalate-supported Ca²⁺ uptake in cardiac homogenates was measured by amodified Millipore filtration technique (Piacentino et al., 2003; Luo etal., 1994; Holemans et al., 2014 and Anderson et al., 2015). Heart,soleus and quadriceps tissues were isolated from WT, Tg and KO mice andrapidly frozen in liquid nitrogen and stored at −80° C. until processed.Frozen tissue samples were homogenized in 50 mM phosphate buffer, pH 7.0containing 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 0.3 mM PMSF and 0.5 mMDTT. Ca²⁺ uptake was measured in reaction solution containing 40 mMimidazole pH 7.0, 95 mM KCl, 5 mM NaN₃, 5 mM MgCl₂, 0.5 mM EGTA, 5 mM K⁺oxalate, 1 μM ruthenium red and various concentrations of CaCl₂ to yield0.02 to 5 μM free Ca²⁺. Homogenates were incubated at 37° C. for 2minutes in the above reaction buffer and the reaction was initiated bythe addition of ATP (final concentration 5 mM). The data were analyzedby nonlinear regression with computer software (GraphPad Software), andthe K_(Ca) values were calculated using an equation for a generalcooperative model for substrate activation. The values for maximal SERCAactivity were taken directly from the experimental data and normalizedfor total protein concentration (μmol/mg protein/min). Statisticalanalyses were performed using an unpaired t-test and data are presentedas mean±SEM.

Contractile Force of Soleus Muscle.

The soleus muscle was dissected free and suspended in an organ bathmaintained at 37° C. One end was tied to a rigid post and the other wasfastened to a force transducer (Myobath, WPI Inc.). Isometriccontractions were elicited by application of brief current pulses of 0.2msec duration at 80 mA (A385 Stimulator; WPI Inc.). Low frequency pulsetrains (<10 Hz) elicited unfused twitch responses and progressivelyhigher stimulation frequencies produced a smooth tetanic contraction.Contraction tests were separated by a 2 minute rest interval to preventfatigue. Muscle contraction was quantified as the peak force and thetime constant for relaxation that was obtained from a single exponentialfit to the decay in force from 50% of the maximum back to baseline. Thebath contained 118 mM NaCl, 4.7 mM KCl, 1.18 mM MgSO₄, 2.5 mM CaCl₂,1.18 mM NaH₂PO₄, 10 mM glucose, 24.8 mM NaHCO₃ and was continuouslybubbled with 95% 02 and 5% CO₂. d-turbocurarine (0.25 μM) was added toprevent muscle activation from nerve fiber endings.

Skeletal Muscle Electroporation.

Flexor digitorum brevis muscles were electroporated with GFP-taggedconstructs and visualized fresh in physiologic buffer using two-photonlaser scanning microscopy as described previously (DiFranco 2009; Nelsonet al., 2013).

Transthoracic Echocardiography.

Cardiac function and heart dimensions were determined by two-dimensionalechocardiography using a Visual Sonics Vevo 2100 Ultrasound (VisualSonics, Canada) on nonanesthetized mice. M-mode tracings were used tomeasure anterior and posterior wall thicknesses at end diastole and endsystole. Left ventricular (LV) internal diameter (LVID) was measured asthe largest anteroposterior diameter in either diastole (LVIDd) orsystole (LVIDs). A single observer blinded to mouse genotypes performedechocardiography and data analysis. Fractional shortening (FS) wascalculated according to the following formula: FS(%)=[(LVIDd−LVIDs)/LVIDd]×100. Ejection fraction (EF %) was calculatedby: EF (%)=EDV−ESV/EDV (ESV; end systolic volume, EDV; end diastolicvolume).

Transmission Electron Microscopy.

Hearts were fixed by perfusion with 4% paraformaldehyde and 1%glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Samples wereprocessed by the University of Texas Southwestern Medical CenterElectron Microscopy Core facility. Briefly, fixed tissues werepost-fixed, stained, dehydrated, and embedded in EMbed-812 resin. Tissuesections were cut and post-stained, and images were acquired on a FEITecnai G² Spirit TEM.

Example 2—Results

Recently, the inventors discovered MLN as a small open reading frame(ORF) hidden in a transcript annotated as a long non-coding RNA (lncRNA)(Anderson et al., 2015). They hypothesized that many transcriptscurrently annotated as lncRNAs may encode small proteins that haveevaded gene annotation attempts and recent proteomic analyses supportthis notion (Slavoff et al., 2013; Frith et al., 2006 and Nelson et al.,2014). To identify potential muscle-specific micropeptides, theinventors searched bioinformatically using PhyloCSF for putative ORFswithin mouse transcripts annotated as lncRNAs (Lin et al., 2011). Amongthese RNAs, they discovered a previously unrecognized muscle-specificRNA containing a potential ORF of 34 codons, which the inventors calledDwarf Open Reading Frame (DWORF) (FIG. 1A and FIG. 5). The DWORF RNAtranscript is annotated as NONCODE lncRNA gene NONMMUG026737 (Xie etal., 2014) in mice and lncRNA gene LOC100507537 in the UCSC human genome(FIG. 6A). With only 34 codons, DWORF is the third smallest full-lengthprotein known to be encoded by the mouse genome.

The mouse DWORF transcript is encoded by 3 exons on chromosome 3 (FIG.6A). The ORF begins in exon 1, which encodes the first four amino acidsof the protein, and the remaining protein is encoded in exon 2.Alternative usage of two adjacent splice acceptor sequences betweenexons 1 and 2 produces two transcripts that differ by a 3 nucleotideinsertion. Based on RNA-seq reads mapping to the exon junction, theshorter isoform of 34 amino acids appears to be substantially moreabundant in the heart. The DWORF ORF is conserved to lamprey, the mostdistant extant vertebrate species for which a genome sequence iscurrently available (FIG. 1A) and the ORF scores positively on PhyloCSF(FIG. 6B). The C-terminal region is enriched in hydrophobic amino acidsand is predicted to encode a tail-anchored transmembrane peptide(Sonnhammer et al., 1998; Krogh et al., 2001 and Goujon et al., 2010).The N-terminal region is less stringently conserved, but most sequences(except for that of Anolis carolensis) contain multiple charged residues(primarily lysine and aspartic acid) in this region. Unless otherwisestated, further studies focused on the mouse homolog.

Northern blot analysis showed that the DWORF mRNA transcript is robustlyexpressed in the heart (FIG. 1B). By quantitative RT-PCR, DWORF RNA wasalso detected in heart and soleus, a postural muscle group of thehindlimb that contains the greatest enrichment of slow-twitch musclefibers in mice (FIG. 1C), as well as diaphragm, which contains someslow-twitch fibers but is primarily a fast-twitch muscle in mice (Guidoet al., 2010 and Schiaffino and Reggiani, 2011). Notably, DWORF was notdetected in the quadriceps, a fast-twitch muscle group, or in cardiacatrial muscle. DWORF is not expressed in the prenatal heart, butgradually increases in abundance post-natally (FIG. 1D).

To verify that the DWORF transcript encodes a protein, the inventorscloned the predicted 5′ untranslated region (UTR) including the firstthirteen codons of the DWORF RNA into a HaloTag expression vector thatcontains a protein tag but lacks a Kozak sequence and start codon.Expression of the empty and DWORF-containing constructs in non-musclecells (COST) followed by western blot analysis with an antibody againstthe HaloTag protein showed that the full-length fusion protein wasexpressed as expected, demonstrating that the DWORF 5′ UTR is capable ofinitiating translation (FIG. 1E). To confirm that the DWORF transcriptencodes a protein, the inventors raised a polyclonal rabbit antibodyagainst the N-terminal 12 amino acids of the predicted mouse DWORFprotein. Western blot analysis revealed a single band at the expectedmolecular weight of 3.8 kDa in soleus and heart, but not in othertissues (FIG. 1F).

Given its abundance in the heart, the inventors examined whether theexpression of DWORF mRNA or protein changed in response to pathologicalcardiac signaling. Indeed, in mice bearing an αMHC-Calcineurin A (CnA)transgene, which serve as a model of hypertrophic heart disease thatprogresses to dilated cardiomyopathy by six months of age (Molkentin etal., 1998), DWORF mRNA was down-regulated in dilated transgenic heartsof six month-old mice (FIG. 1G). The level of DWORF protein was evenmore dramatically down-regulated in these hearts (FIG. 1H). DWORF mRNAwas also down-regulated in ischemic failing human hearts, linkingchanges in DWORF expression with human cardiomyopathies (FIG. 1I)(Makarewich et al., 2015).

The inventors investigated the subcellular distribution of DWORF inskeletal muscle fibers by electroporation of a GFP-DWORF expressionvector into the flexor digitorum brevis muscle of the mouse foot (Nelsonet al., 2013). Live imaging analysis using two-photon excitationmicroscopy to simultaneously visualize GFP and myosin (using secondharmonic generation) showed that GFP-DWORF localized in an alternatingpattern with myosin (FIG. 2A), a distribution resembling the location ofthe SR. GFP-SLN and GFP-PLN were individually expressed in the flexordigitorum brevis muscle for comparison. The visible co-localization ofGFP-DWORF, GFP-SLN and GFP-PLN was striking, demonstrating notabletransverse and lengthwise striations typical of longitudinal SR. Thesubcellular distribution of GFP-DWORF in transfected COS7 cells alsooverlapped with that of mCherry-SERCA1 in the endoplasmic reticulum (ER)and peri-nuclear regions (FIG. 2B).

Because GFP-DWORF co-localizes to the SR with SERCA, the inventorstested whether the two proteins physically interact. COS7 cells wereco-transfected with GFP or GFP-DWORF and Myc-tagged SERCA1, 2a, 2b, 3a,or 3b. Immunoprecipitation with a GFP antibody co-precipitated GFP-DWORFwith all isoforms of SERCA, but did not pull down SERCA in GFPtransfected samples lacking DWORF (FIG. 2C). The inventors next examinedwhether co-expression of DWORF with SERCA would affect complex formationbetween SERCA and PLN, SLN, or MLN. Indeed, they observed a reduction inthe binding of HA-PLN, -SLN, and -MLN with SERCA when co-expressed withGFP-DWORF (FIG. 2D and FIG. 7), suggesting that binding of DWORF and PLNto SERCA are mutually exclusive. Co-expression of Myc-SERCA2a withvarious ratios of GFP-DWORF and GFP-PLN followed by immunoprecipitationwith anti-Myc and immunoblotting with anti-GFP indicated that DWORF andPLN have similar binding affinities for SERCA (FIG. 2E).

To assess the functions of DWORF in vivo, the inventors generated mousemodels of gain and loss of DWORF function. DWORF over-expression in theheart was achieved by expressing untagged DWORF under the control of thecardiomyocyte-specific α-myosin heavy chain (αMHC) promoter intransgenic mice. Two transgenic founders that overexpressed the proteinwere selected for further studies. Other proteins involved in Ca²⁺handling were largely unaffected in these transgenic mice (FIGS. 8A-C).

The inventors used the CRISPR/Cas9 system to disrupt the coding frame ofthe DWORF protein in mice. A single guide RNA (gRNA) was designed totarget the coding sequence of exon 2 before the transmembrane region(FIG. 3A). F₀ pups were screened for indels and a founder with atwo-base-pair insertion that disrupts the ORF after codon 16 was chosenfor further analysis. Breeding of heterozygous DWORF knockout (KO) miceyielded homozygous mutant offspring at expected Mendelian ratios. DWORFKO mice were phenotypically normal and displayed no obvious phenotype upto 1 year of age. Western blots of ventricular and soleus muscle probedwith anti-DWORF antibody showed that the DWORF protein was completelyeliminated in muscle tissues of homozygous mutant mice (FIG. 3B).Surprisingly, the DWORF transcript was up-regulated ˜4-fold in the DWORFKO tissue (FIG. 9A), suggesting a potential feedback mechanism toenhance DWORF expression. Several notable RNA transcripts were notchanged in DWORF KO mice, including those encoding the Ca²⁺-handlingproteins SERCA2 and PLN and the cardiac stress markers Myh7 and atrialnatriuretic peptide (Nppa). Western blot analysis of heart (FIG. 9B) andsoleus muscle (FIG. 9C) homogenates revealed no detectable changes inprotein expression level or phosphorylation state of major Ca²⁺-handlingproteins.

The inventors examined whether Ca²⁺ flux was altered in adultcardiomyocytes from WT, αMHC-DWORF Tg and DWORF KO mice using thefluorescent Ca²⁺ indicator dye, fluo-4. Isolated cardiomyocytes wereloaded with fluo-4, mounted on a temperature controlled perfusionchamber, and electrically stimulated at 0.5 Hz to initiate intracellularCa²⁺ transients which were monitored by epifluorescence. Peak systolicCa²⁺ transient amplitude and SR Ca²⁺ load were significantly increasedin Tg myocytes (FIGS. 3C-D). The pacing-induced Ca²⁺ transient decayrate was also significantly enhanced in the Tg myocytes (FIG. 3E),suggesting that SERCA is more active in these cells. The decay rate ofcaffeine-induced Ca²⁺ transients was unchanged in Tg myocytes, whichindicates that the activity of the Na⁺/Ca²⁺ exchanger (NCX) is notaltered (FIG. 3F). Tg myocytes had higher baseline measurements ofcontractility as measured by fractional shortening (FIGS. 3G-H), peakCa²⁺ transient amplitude (FIG. 3I), and Ca²⁺ transient decay rate (FIG.3J) and responded less strongly to beta-adrenergic stimulation byisoproterenol, likely because they are functioning at close to maximallyactive levels under baseline conditions. In the absence of increasedprotein abundance of SERCA or changes in other known Ca²⁺ handlingproteins, these findings indicate that SERCA activity is increased inmuscle cells over-expressing DWORF.

The effect of DWORF ablation on skeletal muscle contractile function wasassessed by measuring twitch force at multiple stimulation frequenciesin isolated soleus muscles from WT and KO mice (Tupling et al., 2011).The inventors did not observe significant differences in peak muscleforce between genotypes and saw no differences in relaxation rates atlow, non-tetanic stimulation frequencies; however, at tetanus-inducingfrequencies, relaxation rates were significantly slowed in DWORF KOmuscles following tetanus (FIG. 3K). The effect on post-tetanicrelaxation times may suggest that DWORF expression is particularlybeneficial for recovery from prolonged contraction and Ca²⁺ release.

Oxalate-supported Ca²⁺-dependent Ca²⁺-uptake measurements in musclehomogenates provide a direct measurement of SERCA enzymatic activity(Tupling et al., 2011 and Davis et al., 1983). The inventors used thistechnique to measure SERCA activity in hearts of WT, Tg and KO mice.Hearts over-expressing DWORF showed an apparent increase in SERCAactivity at lower concentrations of Ca²⁺ substrate in both of theinventors' transgenic lines quantified as a higher affinity of SERCA forCa²⁺ (reduction in K_(Ca)) and DWORF KO hearts exhibited a less obviousbut still significant decrease in the affinity of SERCA for Ca²⁺ asindicated by an increase in K_(Ca) (FIG. 4A, FIGS. 10A-B, and Table S1)The inventors did not observe changes in the maximal rate of Ca²⁺ pumpactivity (V_(max)) in any of the inventors' genotypes (Table S1).Because DWORF is most abundant in the slow-twitch soleus muscle group,they also measured SERCA activity in soleus homogenates from WT and KOmice and used quadriceps muscles as a control, since DWORF is notexpressed in this muscle group. The soleus muscle of DWORF KO miceshowed a decreased apparent affinity for Ca²⁺ compared to WT muscles(FIG. 4B and Table S2). These differences were not observed inquadriceps muscle (FIG. 9C and Table S3).

Based on gain and loss of function studies, these results demonstratethat DWORF enhances SR Ca²⁺ uptake and myocyte contractility (FIG. 4C).Because DWORF can displace the inhibitory peptides PLN, MLN and SLN fromSERCA, the inventors speculate that this mechanism represents the basisfor its stimulatory influence on SERCA. However, it is also conceivablethat DWORF acts through additional mechanisms and that the changes theinventors observed in Ca²⁺ dynamics are secondary to some other processthat DWORF regulates. Because DWORF increases the activity of the SERCApump, it represents an attractive means of enhancing cardiaccontractility in settings of heart disease. These findings show thatcardiac-specific overexpression of DWORF (DWORF TG) in a mouse model ofdilated cardiomyopathy (Muscle LIM protein knockout mice, (MLP KO))leads to an enhancement of SERCA activity and a profound rescue of theheart failure phenotype seen in these animals (FIG. 13-15). Finally,these results underscore the likelihood that many transcripts currentlyannotated as non-coding RNAs encode micropeptides with importantbiological functions and that these micropeptides may have evolved assingular protein domains that exert their function by directlymodulating the activities of larger regulatory proteins

TABLE S1 Ca²⁺-dependent Ca²⁺-uptake assay K_(Ca) and V_(max) values intotal heart homogenates Genotype K_(Ca) (μM) p-value V_(max)(nmol/mg/min) p-value WT 0.2038 ± 0.0067 NA  62.81 ± 10.26 NA Tg Line 10.1670 ± 0.0072 0.0013 64.59 ± 5.81 0.824 Tg Line 2 0.1810 ± 0.00780.0279 63.25 ± 9.34 0.887 KO 0.2328 ± 0.0110 0.0478 66.15 ± 5.77 0.687

TABLE S2 Ca²⁺-dependent Ca²⁺-uptake assay K_(Ca) and V_(max) values intotal soleus muscle homogenates Genotype K_(Ca) (μM) p-value V_(max)(nmol/mg/min) p-value WT 0.1663 ± 0.0058 NA 16.07 ± 1.33 NA KO 0.1923 ±0.0086 0.0478 14.72 ± 1.84 0.688

TABLE S3 Ca²⁺-dependent Ca²⁺-uptake assay K_(Ca) and V_(max) values intotal quadriceps homogenates Genotype K_(Ca) (μM) p-value V_(max)(nmol/mg/min) p-value WT 0.1782 ± 0.0119 NA 44.64 ± 4.83 NA KO 0.1809 ±0.0047 0.8446 48.98 ± 3.91 0.511

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this disclosure havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the disclosure as defined by theappended claims.

VI. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of promoting the activity of the SERCAcalcium pump in a cell comprising contacting said SERCA pump with DWORF.2. The method of claim 1, wherein DWORF has the sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO:
 9. 3. Themethod of claim 1, wherein DWORF is linked to a heterologous peptide orpolypeptide segment.
 4. The method of claim 3, wherein the heterologouspeptide or polypeptide segment is HIV TAT.
 5. The method of claim 1,wherein DWORF is contacted as a peptide or polypeptide.
 6. The method ofclaim 1, wherein DWORF is contacted by expression of a nucleic acidsegment coding for DWORF or a functional fragment thereof, said nucleicacid segment being under the control of a promoter active in eukaryoticcells.
 7. The method of claim 6, wherein said nucleic acid segment isprovided as naked DNA or modified mRNA.
 8. The method of claim 6,wherein said nucleic acid segment is provided in a viral particle. 9.The method of claim 6, wherein said nucleic acid segment is provided asa non-viral expression construct in a nanoparticle, microparticle orlipid vehicle.
 10. The method of claim 1, wherein further comprisingcontacting the SERCA calcium pump with a second SERCA activating agent.11. The method of claim 10, wherein the second SERCA activating agent isistaroxime.
 12. The method of claim 1, wherein said cell is located in aliving mammal.
 13. The method of claim 12, wherein living mammal is anon-human mammal.
 14. The method claim 12, wherein the living mammal isa human.
 15. The method of claim 12, wherein contacting occurs at leasta second time.
 16. The method of claim 12, wherein contacting occurs ona chronic basis.
 17. The method of claim 12, wherein DWORF is providedto said mammal intravenously, intradermally, intraarterially,intraperitoneally, intranasally, topically, intramuscularly,subcutaneously, mucosally, intrapericardially, intraumbilically, orally,via injection, via infusion, via continuous infusion, via a catheter,via a lavage, in creams, or in lipid compositions (e.g., liposomes). 18.The method of claim 12, wherein said mammal suffers from a disordercharacterized by or comprised of cytosolic calcium overload.
 19. Themethod of claim 18, wherein said cytosolic calcium overload condition isselected from the group consisting of heart failure, restenosis andmuscular dystrophy.
 20. The method of claim 19, further comprisingadministering to said mammal a second therapy for heart failure,restenosis or muscular dystrophy.
 21. An isolated polypeptide comprisinga sequence selected from the group consisting of wherein DWORF has thesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 andSEQ ID NO:
 9. 22. The isolated polypeptide of claim 21, wherein saidisolated polypeptide is disposed in a pharmaceutically acceptablebuffer, diluent or excipient.
 23. The isolated polypeptide of claim 21,wherein said isolated polypeptide consists essentially of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO:
 9. 24. Theisolated peptide of claim 21, wherein said isolated polypeptide consistsof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO:9.
 25. The isolated polypeptide of claim 21, wherein said isolatedpolypeptide is linked to a heterologous peptide or polypeptide segment.26. An isolated nucleic acid segment encoding a polypeptide comprising asequence selected from the group consisting of wherein DWORF has thesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 andSEQ ID NO:
 9. 27. The isolated nucleic acid segment of claim 26, whereinsaid isolated nucleic acid segment is disposed in a pharmaceuticallyacceptable buffer, diluent or excipient.
 28. The isolated nucleic acidsegment of claim 26, wherein said isolated nucleic acid segment encodesa polypeptide consisting essentially of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7 and SEQ ID NO:
 9. 29. The isolated nucleic acidsegment of claim 26, wherein said isolated nucleic acid segment encodesa polypeptide consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7 and SEQ ID NO:
 9. 30. The isolated nucleic acid segment ofclaim 26, wherein said isolated nucleic acid segment is linked to aheterologous nucleic acid segment.
 31. The isolated nucleic acid segmentof claim 30, wherein said heterologous nucleic acid segment is a cellpermeability peptide, such as HIV TAT.
 32. The isolated nucleic acidsegment of claim 30, wherein said heterologous nucleic acid segment is apromoter.
 33. The isolated nucleic acid segment of claim 30, whereinsaid heterologous nucleic acid segment is an expression construct. 34.The isolated nucleic acid segment of claim 33, wherein said expressionconstruct is a viral expression construct, such as an adenovirusconstruct, a retrovirus construct, a pox virus construct, or aherpesvirus construct.
 35. The isolated nucleic acid segment of claim32, wherein said expression construct is a non-viral expressionconstruct.